Aspects of 1,25-dihydroxyvitamin D3 (1,25-D3) receptor dynamics will be studied in chickens and rats to gain insight concerning the molecular mechanism of 1,25-D3 action in bone, intestine and pituitary. First we will examine critically the localization of receptors with regard to the animal's vitamin D status. To monitor physical and/or chemical changes for unoccupied and occupied receptors, direct labeling or exchange labeling assay techniques will be utilized. We will also ascertain whether or not there is heterogeneity for these hormone binding sites within the same tissue as well as between several target tissues. Since alterations in the rates of synthesis and degradation of receptor could affect markedly the responsiveness to 1,25-D3, studies will be conducted utilizing protein synthesis inhibitors or a "density shift" approach to examine the fate of receptors in the presence or absence of hormone. An attractive and widely held hypotheses is that these receptors bind to some nuclear component. Differential solubilization with salt, DNase I, and intercalating drugs will be employed to determine if receptors are entirely chromatin associated. Determination of nucleosome binding will be carried out by micrococcal nuclease digestion and sucrose density centrifugation. To compliment our receptor-chromatin studies, we will investigate whether histones modulate 1,25-D3-receptor-DNA binding interactions. This will be accomplished through the use of immobilized DNA and histones in a competitive binding assay. Analysis of limited proteolytic digestion of 1,25-D3 receptors will also be conducted to ascertain whether the sterol, DNA, and histone binding sites are topographically distinct domains of the receptor. Studies will also be initiated to define more precisely the temperal relationship between 1,25-D3 receptor binding and induction of responses. To accomplish this, primary cultures of pituitary cells or GH3 colonal pituitary cells will be used to study 1,25-D3 induction of prolactin synthesis. Also, a cDNA probe to rat prolactin will be employed to determine if 1,25-D3 can directly affect gene expression. Advances that promise further elucidation of receptor dynamics will hopefully further our understanding as to whether there is a common denominator(s) for 1,25-D3 action and to enable us to dissect out information where responses might diverge.